Effects Of Thyroid Hormone On Mucin 5AC Expression And Its Mechanism In Pulmonary A549 Cell Line

Objective:To explore the effects of thyroid hormone on mucin(MUC)5AC expression in the airway epithelial cells and its mechanism.Methods:Expression of MUC5AC in A549 cells:The well cultured airway epithelial cells of the line A549 were exposed to serial dilutions of thyroid hormone for 24 hours,the levels of MUC5AC protein secreted into the supernatants were determined by ELISA.The expression of MUC5AC in A549 cells was detected with RT-PCR on transcript levels.Design and construction of luciferase reporter gene plasmids containing different length of human MUC5AC gene promoter:Using the restriction enzymes,PCR and gene recombination techniques,four luciferase reporter gene plasmids containing different length of human MUC5AC gene promoter were constructed.The plasmids and intracontrol plasmid pRL-TK were co-transfected into the human pulmonary A549 cells with eukaryotic gene transfected techniques,and the relative luciferase activities were detected in the transfected A549 cells. Results:Expression of MUC5AC in A549 cells:The expression of MUC5AC protein was decreased significantly in thyroid hormone treated groups compared with the without thyroid hormone treated.The expression of MUC5AC protein in different dose were lower than that of the control group.Thyroid hormone decreased MUC5AC mRNA levels in A549 cells.Construction of luciferase reporter gene plasmids containing different length of human MUC5AC gene promoter:Restriction enzyme digestion and DNA sequencing were performed to confirm the successful construction of four luciferase reporter gene plasmids containing different sequences of human MUC5AC promoter. Thyroid hormone could decrease the expression of luciferase reporter gene plasmids containing -1330bp and -1250bp version of MUC5AC promoter in transfected A549 cells,but could not decrease the the expression of luciferase reporter gene plasmids containing -1200bp and -1020bp version of MUC5AC promoter.Conclusions:1.Thyroid hormone can decrease the level of MUC5AC protein and mRNA respectively in the airway epithelial cells A549 by ELISA and RT-PCR.2.We constructed a series of luciferase reporter gene plasmids containing different length of human MUC5AC gene promoter by gene recombination and PCR.The constructed luciferase reporter gene plasmids include pGL3-MUC5AC-1330/+48,pGL3-MUC5AC-1250/+48,pGL3-MUC5AC-1200/+48, pGL3-MUC5AC-1020/+48.3.After above luciferase reporter plasmids and intracontrol plasmids pRL-TK were co-transfected into A549 cells,it is observed that thyroid hormone could decrease the expression of luciferase reporter gene plasmids containing -1330bp and -1250bp of MUC5AC promoter in the transfected A549 cells,whereas thyroid hormone could not decrease the expression of luciferase reporter gene plasmids containing -1200bp and -1020bp of MUC5AC promoter.These results suggest that there are thyroid hormone-responsive region between -1250bp and -1200bp of promoter.